Regulation of glycolysis and gluconeogenesis, and the mechanism of anti-diabetic drugs

Prof. A. P. Halestrap

ReferencesPilkis, S. J. & Granner, D. K. (1992). Molecular physiology of the regulation of hepatic

gluconeogenesis and glycolysis. Annu. Rev. Physiol. 54 885-909.

Van Schaftingen, E. (1993). Glycolysis Revisited. Diabetologia 36, 581-588.

Moller, D. E. (2001) New drug targets for type 2 diabetes and the metabolic syndrome. Nature 414: 821-827

Rutter, G. A.; Xavier, G. D., and Leclerc, I. Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochemical Journal. 2003; 3751-16

Gluconeogenesis

De novo synthesis of glucose as opposed to glycogenolysisWhat

Liver and proximal convoluted tubules of the kidney (late in starvation - pH regulation in acidosis involves conversion of glutamine to ammonia (excreted) and 2-oxoglutarate which forms glucose by gluconeogenesis)

Where

NH4+ NH4

+

CO2

CO2

HCO3-

H+

H+ Na+

Na+Glutamine Glutamate 2-Oxoglutarate Glucose

NH3 NH3

NH3 NH3

BLOOD

URINE

Liver proximal tubule epithelial cell

GlucoseGlutamine

Glutaminase Glutamate DH GNG

After exercise, starvation, diabetes, at birth.When

Lactic acid (exercise / Cori cycle)

Substrates

Fructose (from sucrose)

Glycerol and propionate (from odd chain fatty acid -oxidation) are the only components of triglycerides that can be used for glucose production.

Alanine Alanine

Amino acids

2-Oxo acids

Glutamate

Urea

Some amino acids and especially alanine and glutamine (alanine cycle and glutamine cycle used to transfer amino groups from muscle to liver for urea synthesis).

2-Oxoglutarate

Pathway reverse of glycolysis except for three steps with very negative G.

Fructose-1,6-bisphosphatase instead of phosphofructokinase

Glucose-6-phosphatase instead of glucokinase (hexokinase)

Gluconeogenesis needs NADH

Gluconeogenesis needs ATP

Pyruvate carboxylase plus phosphoenolpyruvate carboxykinase (PEPCK) instead of pyruvate kinase.

HCO3- Uses 2 ATPs to reverse a glycolytic step that makes 1 ATP

Glutamate Glutamate

2-oxoglutarate 2-oxoglutarate

Note that pyruvate carboxylation is mitochondrial whereas PEPCK is cytosolic; hence we need oxaloacetate to cross mitochondrial inner membrane.

Where L-lactate is the substrate this occurs as aspartate since lactate conversion to pyruvate produces NADH to drive glycolysis backwards (Route 1 in diagram).

For most substrates oxaloacetate crosses as malate and effectively transfers NADH from the mitochondria (where it is abundant from fatty acid oxidation and citric acid cycle activity) to the cytosol (Route 2)

Pyruvate carboxylase in mitochondria

Cytosol

Regulation can be:Long term (e.g. starvation and diabetes)

Long and medium term regulation involve changes in gene expression whilst short term regulation involves a change in enzyme activity or substrate supply.

Note that both long and short term regulation involves the those enzymes that can participate in futile cycles.

Short term (e.g. during and after exercise and other stresses - Cori cycle).

Medium term (birth and acidosis)

Regulation

# By regulator protein. Note also that pyruvate carboxylase is regulated by allosteric effectors and substrate supply

#

Primarily mediated through an increased glucagon/insulin ratio causing induction of gluconeogenic enzymes (especially PEPCK, but also other key GNG enzymes in Table 1) with permissive effect of glucocorticoids such as cortisol. Glycolytic enzymes such as GK and PK are repressed.

Starvation and Diabetes both induce a large decrease in glucagon / insulin ratio and cause a 5-10 fold increase in PEPCK in liver and 2-3 fold increase in kidney. In kidney PEPCK induction also occurs in response to acidosis.

In the liver it can be shown that PEPCK protein synthesis induced by glucagon follows a rise in cyclic AMP and mRNAPEPCK synthesis.

After 20 min mRNA increased 5-fold: After 90 min 9-fold)

mRNA degradation is not affected (addition of -amanitin to block RNA synthesis promotes the same rate of PEPCK degradation in controls and glucagon- treated livers).

The mechanism involves a range of regulatory elements in the PEPCK promoter including cAMP, gluocorticoid and thyroid hormone response elements. (Other promoters have similar regulatory elements).

Long and medium term regulation

Thyroid hormone response element

Glucocorticoid response element cAMP response element

Note the immense increase in PEPCK activity seen at birth are also brought about by large changes in glucagons/insulin ratios. Transgenic mice in which the PEPCK promoter is linked to the growth hormone gene greatly enhances the production of growth hormone at birth, leading to very large mice that grow at twice normal rate!

GHPEPCK

This involves both substrate supply and hormones.

Short term regulation

Note that alcohol reduces gluconeogenesis by increasing NADH/NAD+ and hence decreasing [oxaloacetate].

Stimulation by glucagon and other hormones that increase cyclic AMP (adrenaline via -receptors in some species) regulate enzyme activity through the activation of protein kinase A.

These effects are antagonised by insulin which lowers cyclic AMP.

Stress hormone including adrenaline (1-receptors), opiates, vasopressin and angiotensin work through activation of phospholipase c

Hormone Receptor PLC activation

PIP2

DAG IP3 Ca2+

Protein kinase C Calmodulin-dependent protein kinases

Mitochondrial metabolism

Identification of control points

1. Effects of hormones on the rates of gluconeogenesis from different substrates

Glucagon and Ca-hormones

Glucagon

2. Futile cycle measurements

Futile cycling only occurs to a significant extent in the fed state and is insignificant in the starved state.

Glucagon inhibits futile-cycling at both PEPCK / PK and PF-1-K / Fru-1,6-Pase whilst Ca-mobilising hormones (e.g.vasopression and -adrenergic agonists) only inhibit futile-cycling at PEPCK / PK and to a lesser extent than glucagon.

3. Crossover plots

Glucagon induced changes in metabolite concentration

LACPYR

MALPEP

3-PGADHA

G3PF16bisP

G6PGluc

0

50

100

150

200

250

100

Me

tab

oli

te l

ev

el

as

% c

on

tro

l

L-Lactate as substrate

DHA as substrate

Crossover

Crossover

Glucagon produces a crossover at both PEPCK / PK and PF-1-K / Fru-1,6-

Pase

-adrenergic agonists only

produce a crossover at

PEPCK / PK step

4. Flux control coefficient measurements

Flux control coefficient x 100

[L-Lactate] 5mM 0.5mM 5mM 0.5mM

These data show that pyruvate carboxylase is the most rate limiting process

Most rate determining

And that regulation by glucagon at both PEPCK / PK and PF-1-K / Fru-1,6-P2ase

Pyruvate transport

Mechanisms of short term regulation of gluconeogenesis

1. Pyruvate to phosphoenolpyruvate step

a) PEPCK Short term regulation is primarily through the supply of oxaloacetate whose cytosolic concentrations are less than the enzymes Km (about 9 M).

There may also be regulation through changes in the concentration of 2-oxoglutarate, a competitive inhibitor. Glucagon and Ca-mobilising hormones decrease the concentration of 2-oxoglutarate by a Ca-mediated activation of 2-oxoglutarate dehydrogenase.

Pathologically, the enzyme is inhibited if tryptophan levels are high. Tryptophan is broken down to quinolinate which chelates Fe2+, an essential cofactor.

COO

COO

Fe2+

b)    Pyruvate kinase The liver isoform of PK is a key regulator of gluconeogenesis in the FED state. It is inhibited by protein kinase A mediated phosphorylation , which decreases the substrate affinity of the enzyme. (The kidney M2 isoform can also be regulated in this way).

[PEP] mM1 2

Act

ivity

PhosphorylationAlanineATP

F16P2

Phosphorylation by calmodulin-dependent protein kinase has a similar but less potent inhibitory effect and accounts for some of the effects of Ca-mobilising hormones on gluconeogenesis.

For glucagons in the fed state, there is a strong correlation between phosphorylation / inhibition of PK and stimulation of gluconeogenesis.

At the levels of glucagon present in the starved state PK is already almost totally inhibited and thus does not play a role in the regulation of gluconeogenesis under these conditions.

d) Pyruvate carboxylase Exclusively mitochondrial enzyme with Km for

pyruvate of about 200M. This is in the physiological range and regulation through substrate supply is important.

PC is critically dependent on acetyl-CoA which acts as an allosteric activator over the physiological range of concentrations, and this provides a regulatory link pyruvate carboxylation to fatty acid oxidation.

[Acetyl CoA] M

250 500

Act

ivity

Physiological range

Enzyme in mitochondria

Fatty acid oxidation

oxidationFatty acid

[Acetyl-CoA]

Cyclic AMP PKA and

CPT1

PC is inhibited by glutamate and by increases in the ADP/ATP ratio. These provide a mechanism by which glucagon and Ca-mobilising hormones can stimulate pyruvate carboxylase.

Stimulation of

Pyruvatecarboxylase

gluconeogenesis

Ca-sensitivedehydrogenases

NADH

NAD

Hormones

Mitochondrial [Ca ]2+

Matrix

Matrix

K entry into+

[PPi]

Matrix

volume Activation ofrespiration

ATPADP

[ 2-OG]

[Glu]Relieve

inhibition of PEPCK

Sites used for inhibiting GNG

Hypoglycaemic agents and antidiabetic drugs

A. Inhibitors of fatty acid oxidation

Inhibitors of carnitine palmitoyl transferase 1, especially cyclo-oxirane derivatives which are activated by fatty-acyl CoA synthetase to their CoA derivative which inhibits CPT1 with Ki values of less than 1M.

O

R COOH

O

R COSCoACoA

ATP AMP + PPi

Cl CH2(CH2)4CH3(CH2)13-

POCA Tetradecylglycidate

Inhibitors of -oxidation such as hypoglycin (unripe ackee fruit - Jamaican vomiting sickness)

CH2 NH2 CH2 O

 

Hypoglycin Transamination

CH2 C CH-CH2-CH-COOH CH2 C CH-CH2-C-COOH

Methylene-cyclopropyl-propionic acid

(Pent-4-enoate has a similar effect)

CH2 C CH-CH2-C-S-CoA

CH2 O

Methylene-cyclopropyl-acetyl-CoA

Irreversible inhibitor of butyryl-CoA dehydrogenase

Oxidative decarboxylation

B. Inhibitors of the respiratory chain

The respiratory chain has a high flux control coefficient for gluconeogenesis

50 1000

Rate of GNGRespiratory chain activity

[ATP]

[Respiratory chain inhibitor]

V / JAlthough [ATP] changes little the calculated ATP/ADP ratio drops a lot and calculated free

[AMP] increases

Thus could mild inhibitors of the respiratory chain are potential anti-diabetic agents? The surprising answer is yes and the most commonly prescribed antidiabetic drug, metformin, probably works this way.

Owen, M. R.; Doran, E., and Halestrap, A. P. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochemical Journal. 2000; 348607-614.

0 100 200 300 400Time (min)

0

20

40

60

80

100

Rat

e o

f re

spir

atio

n (

% o

f co

ntr

ol)

Succinate

Glutamate + malate

Incubation with 10mM metformin at 8oC

The diabetic drugs metformin and phenformin (biguanides) act on the respiratory chain.

Metformin

N-C-NH2

NHCH3

CH3

+2

0 2.5 5 7.5 10

[Metformin] (mM)

0

20

40

60

80

100

Res

pir

ato

ry r

ate

as %

of

con

tro

l

0 0.1 0.2 0.3 0.4 [Phenformin] (mM)

K0.5 14.9 ± 1.19 mM

K0.5 0.05 ± 0.0015 mM

Incubation at 8oC with inhibitor for 4 hr

(metformin) or 5 min (phenformin)

Phenformin

N-C-NH2

NH

CH3

CH2

+2

Phenformin

Metformin

0 10 20 30 40 50

[Metformin] (mM)

0

20

40

60

80

100

Res

pir

ato

ry r

ate

as %

of

con

tro

l

0 2 4 6 8 10

[Phenformin] (mM)

K0.5 79.0 ± 3.4 mM

K0.5 2.23 ± 0.18 mM

Metformin inhibits immediately in sub-mitochondrial particles but requires higher concentrations

Cf 15 mM in intact energised mitochondria

Cf 0.05 mM

Metformin

= -180mV

Accumulation

Positive charge allows slow accumulation in mitochondria where they act as weak inhibitors of complex 1.

Uptake is self-limiting: if excessive inhibition occurs drops preventing further accumulation.

Phenformin is much more potent than Metformin because it is more hydrophobic and enter the mitochondria more rapidly. It has a much higher risk of causing the rare side-effect of severe lactic acidosis.

N-C-NH2

NHCH3

CH3

+2

50

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90

100

110

120

130

Sta

te 3

re

sp

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tio

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as

% C

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Glutamate / malate Succinate

[Metformin] 50M 100M 50M 100M

(5)*

(4)* (4)**

(5)*

(4)

(4)

(3)

(4)

24 hours

60 hours

Prolonged exposure allows metformin to inhibit the respiratory chain at therapeutic doses

Hepatoma cell incubated with metformin for the time shown and then mitochondrial respiration measured in permeabilised cells.

0 50 100 150 200

0

150

300

450

600

750

900

Control

Glu

co

se

pro

du

cti

on

(n

mo

le/m

g)

Metformin

Time (min)

2 mM Metformin

5 mM Metformin

45 90 1500

20

40

60

80

100

Pe

rce

nta

ge

in

hib

itio

no

f g

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5/1

36

/9

Time of incubation (min)

2mM Metformin5mM Metformin

Time dependent inhibition of gluconeogenesis in rat liver cells by metformin

Direct effects of metformin on GNG via changes in ATP/ADP ratio and NADH/NAD+ ratio

Inhibition of respiration

NADH

NAD

ATP

ADP

[Lactate]

[Pyruvate]

Pyruvate

Biguanides

and fatty acid oxidation

[Acetyl-CoA]

carboxylase

Inhibition ofgluconeogenesis

The evidence for the proposed mechanism of action comes from measurements of metabolite levels in hepatocytes and whole animals treated with metformin, and from studies on isolated mitochondria.

[Triose phosphates]

[2- + 3-PGA]

[PEP]

Pyruvatekinase

Recent data from several labs has shown that metformin treatment activates AMP dependent protein kinase (AMPK, and that this may play a key role in its anti-diabetic effects. (AMPK inhibitor blocks effects but not very specific). Activation of AMPK is through an indirect mechanism - (no effect on isolated AMPK).

AMPKK

(AMPK Kinase)

AMPKAMPK-P

(Active)

Phosphorylation of target proteins

Inhibition of the respiratory chain [AMP] Metformin

Metformin increases the calculated free [AMP] which could account for this but no increase in total [AMP] can be measured.

LKB1 tumour supressor

? Metformin?

Metformin fails to activate AMPK in cells from an LKB1 knockout mouse

Either total [AMP] measurements mask changes in free [AMP] (quite likely) or metformin acts via some unidentified mechanism.

Zhou, G et al. (2001) Role of AMP-activated protein kinase in mechanism of metformin action J Clin. Invest. 108: 1167-1174. Also papers from Grahame Hardie’s group

AMPK activation can account for effects on metformin on gene transcription (down regulation of fatty acid oxidation and gluconeogenesis genes) and glucose transporter (GLUT-4) up-regulation (expression and translocation) in muscle. Inhibition of acetyl-CoA carboxylase in liver also occurs by this mechanism and may help explain the decrease in plasma free fatty acids and triglycerides.

SREBP-1c (Sterol Response Element Protein)– an important insulin stimulated transcription factor implicated in the pathogenesis of insulin resistance

?Inhibition of the

respiratory chain

[AMP] [ATP]/[ADP]

AMPK may also phosphorylate IRS-1 leading to increased insulin sensitivity

Problems with the AMPK activation theory

Some of the enzyme activities modulated through changed gene expression (e.g. fatty acid synthetase and liver pyruvate kinase) or direct phosphorylation (acetyl CoA carboxylase) are in the opposite direction to insulin.

Many experiments have been performed at concentration of metformin and phenformin far in excess of those used to treat Diabetes

Note that the liver is exposed to much higher [Metformin] than other tissues (except the gut) since it receives the drug from the gut via the portal blood supply. This may be why ingestion of metformin is without major side-effects on tissues such as the heart and brain that are highly dependent on an active respiratory chain.

Sulphonylureas stimulate insulin secretion

Ca 2+

OGlucose K+

Pyruvate

Insulin

mitochondrion

[ATP]

Inhibition of potassium efflux

causes depolarisation and calcium entry

sulphonylureasglyburide = glibenclamide

D. Insulin SensitizersThiazolidinediones such as ciglitazone act as insulin sensitizers, reducing the peripheral insulin resistance that occurs in type 2 diabetes. They are agonists of the peroxisome proliferatory-activated receptor (PPAR), an orphan member of the nuclear hormone receptor superfamily that is expressed at high levels in adipocytes.

PPAR is a central regulator of adipocyte gene expression and differentiation one of whose effects is to decrease Resistin secretion. Resistin works in opposition to leptin and increases insulin resistance (Nature 2001 Jan 18;409(6818):307-12)

Acrp30 is adiponectin PDK4 is PDH kinase 4

Moller, D. E. (2001) New drug targets for type 2 diabetes and the metabolic syndrome. Nature 414: 821-827

Mechanisms of short term regulation of gluconeogenesis

Key regulation is by fructose 2,6-bisphosphate (F-2,6-bisPase). Activates , phosphofructokinase 1 (PFK1) and inhibits fructose-1,6-bisphosphatase F-1,6-bisPase.

Fructose-6-P

Fructose-2,6-bisP

ATP

ADP

Pi

F-2,6-bisPase

Enzyme is 49kDa dimer with both activities on the

same polypeptide

PiInhibited bycitrate and

PEP

Inhibited byF-6-P

(Activates PFK1 and inhibits F-1,6-bisPase)

2. Phosphofructokinase / Fructose-1,6-bisphosphatase step

PFK2Activity switches depending on its

phosphorylation state

Pi

ATP ADPcAMPGlucagon PKA

P

Glucagon [F-2,6-bisP] hence stimulating F-1,6-bisPase and inhibiting PFK1.

Calmodulin-dependent protein kinase does not phosphorylate the enzyme, accounting for the lack of effect of Ca-mobilising hormones on this step.

3.     Glucose-6-phosphatase / glucokinase

Glucose-6-phosphatase (G-6-Pase) is a microsomal enzyme that is induced in starvation and diabetes but for which there is no good evidence for short-term regulation.

Deficiency of G-6-Pase causes glycogen storage disease (Von Gierke’s Disease) since the elevation of G-6-P in the liver inhibits glycogen phosphorylase leading to massive glycogen accumulation in the liver (which is enlarged).

Mutations in any of the G-6-Pase constituent proteins have been shown to produce the disease.

Patients also show severe hypoglycaemia after a short fast because they cannot mobilize their liver glycogen which represents the first source of blood glucose on starvation

Glycogen storage diseases

Repressed in starvation and diabetes.

Short term regulation by fructose which stimulates the conversion of glucose to glucose-6-P in isolated hepatocytes by about 2-4 fold in a reversible fashion.

Glucokinase (GK)

In crude cytosolic extracts of liver F-1P activates GK and F-6P inhibits.

Effect was lost on purification but sensitivity to inhibition by F-6P restored upon addition of an ancillary inhibitory protein (68kDa)

[F-6P] M50 100

GK

Act

ivity

No regulatory protein

With regulatory protein + 200M F-1P

With regulatory protein

Van Schaftingen - the effect correlated with an increase in tissue [Fructose-1-P] and a decrease in [Fructose-6-P].

Note that some individuals have GK deficiency and show early onset and severe Type 2 diabetes.

Regulatory protein resides in the nucleus where GK is also sequestered.

GKR

F-6P

Inactive

R’ R

GK

R’

F-1P

F-1P

R

F-6P

F-6P

Active

Active GK is released from the regulatory protein in response to F-1P or glucose (by some ill-defined

mechanism,) and translocated to the cytosol